导读
宏转录组数据分析第一步。
一、KneadData下载、安装、质控
链接：https://bitbucket.org/biobakery/kneaddata/wiki/Home#markdown-header-installation
wget -c https://files.pythonhosted.org/packages/a4/6a/4176eee7a83b80ac12ca6727df6cb9dd3fec2051cca8a707ccbebc5962d3/kneaddata-0.7.3.tar.gz
# 下载
tar -zxvf kneaddata-0.7.3.tar.gz
# 解压
rm kneaddata-0.7.3.tar.gz
# 删除安装包
python setup.py install
# 安装依赖Trimmomatic和Bowtie2。使用"--bypass-dependencies-install"可取消。
for i in `ls 1.rawdata`; do
/[route]/kneaddata-0.7.3/kneaddata/knead_data.py \
-i 1.rawdata/$i/${i}_1.fq.gz \
-i 1.rawdata/$i/${i}_2.fq.gz \
-o result/qc/kneaddata \
-db /[route]/Databases/hg38 \
--trimmomatic /[route]/Trimmomatic-0.39 \
-t $threads \
--trimmomatic-options "SLIDINGWINDOW:4:20 MINLEN:50" \
--bowtie2-options "--very-sensitive --dovetail --al-gz" \
--remove-intermediate-output \
--run-fastqc-start \
--run-fastqc-end
done
# 质控、去宿主
二、SortMeRNA安装
链接：https://bioinfo.lifl.fr/RNA/sortmerna/
1 apt安装
SortMeRNA version 2.0, 29/11/2014
一键下载、安装、apt安装版本低不推荐
sudo apt install sortmerna
sortmerna --version
2 conda安装
conda create -n python3.6 python=3.6
conda activate python3.6
conda config --show channels
conda install sortmerna
sortmerna --version
# SortMeRNA version 4.2.0
# Build Date: Mar 12 2020
三、建库、去rRNA（only for version2）
version4以上直接用fasta序列，无需建库，非常的奈斯
wget -c http://bioinfo.lifl.fr/RNA/sortmerna/code/sortmerna-2.1-linux-64-multithread.tar.gz
# 在linux没find到数据库，所以再下载一次，里面包含需要的数据库。
indexdb_rna --ref \
./rRNA_databases/silva-bac-16s-id90.fasta,./index/silva-bac-16s-db:\
./rRNA_databases/silva-bac-23s-id98.fasta,./index/silva-bac-23s-db:\
./rRNA_databases/silva-arc-16s-id95.fasta,./index/silva-arc-16s-db:\
./rRNA_databases/silva-arc-23s-id98.fasta,./index/silva-arc-23s-db:\
./rRNA_databases/silva-euk-18s-id95.fasta,./index/silva-euk-18s-db:\
./rRNA_databases/silva-euk-28s-id98.fasta,./index/silva-euk-28s:\
./rRNA_databases/rfam-5s-database-id98.fasta,./index/rfam-5s-db:\
./rRNA_databases/rfam-5.8s-database-id98.fasta,./index/rfam-5.8s-db
# 建索引
# version 2
for i in /[route]/*kneaddata_paired_[12].fastq; do
base=${i##*/}
head=${base%%_*}
tail=${base#*kneaddata_}
sortmerna --ref /[route]/sortmerna-2.1b/rRNA_databases/silva-bac-16s-id90.fasta,/[route]/sortmerna-2.1b/index/silva-bac-16s-db:\
/[route]/sortmerna-2.1b/rRNA_databases/silva-bac-23s-id98.fasta,/[route]/sortmerna-2.1b/index/silva-bac-23s-db:\
/[route]/sortmerna-2.1b/rRNA_databases/silva-arc-16s-id95.fasta,/[route]/sortmerna-2.1b/index/silva-arc-16s-db:\
/[route]/sortmerna-2.1b/rRNA_databases/silva-arc-23s-id98.fasta,/[route]/sortmerna-2.1b/index/silva-arc-23s-db:\
/[route]/sortmerna-2.1b/rRNA_databases/silva-euk-18s-id95.fasta,/[route]/sortmerna-2.1b/index/silva-euk-18s-db:\
/[route]/sortmerna-2.1b/rRNA_databases/silva-euk-28s-id98.fasta,/[route]/sortmerna-2.1b/index/silva-euk-28s:\
/[route]/sortmerna-2.1b/rRNA_databases/rfam-5s-database-id98.fasta,/[route]/sortmerna-2.1b/index/rfam-5s-db:\
/[route]/sortmerna-2.1b/rRNA_databases/rfam-5.8s-database-id98.fasta,/[route]/sortmerna-2.1b/index/rfam-5.8s-db \
--reads $i \
--aligned result/qc/sortmerna/${head}_${tail}.rRNA \
--sam --num_alignments 1 --fastx -a $threads \
--other result/qc/sortmerna/${head}_${tail}.non.rRNA --log -v
done
四、去rRNA（for newest version4）
# version4
route_db="/home/cheng/huty/softwares/sortmerna-2.1b/rRNA_databases"
route_index="/home/cheng/huty/softwares/sortmerna-2.1b/index"
threads=52
mkdir non_rrna
sortmerna \
--ref $route_db/silva-bac-16s-id90.fasta \
$route_db/silva-bac-23s-id98.fasta \
$route_db/silva-arc-16s-id95.fasta \
$route_db/silva-arc-23s-id98.fasta \
$route_db/silva-euk-18s-id95.fasta \
$route_db/silva-euk-28s-id98.fasta \
$route_db/rfam-5s-database-id98.fasta \
$route_db/rfam-5.8s-database-id98.fasta \
--reads CONT1_R1.fastq \
--reads CONT1_R2.fastq \
--fastx \
--paired_out \
--threads $threads \
-v \
--out2 \
--workdir run \
--other non_rrna/cont1
rm -r run # 删除中间文件
# 对kneaddata的结果进行去RNA
参数
--fastx 【布尔】输出fastq
--paired_out 【布尔】输出配对结果
-v 【布尔】不罗嗦
--out2 【布尔】输出结果文件为两个
--workdir dir 中间文件输出目录
--other dir/prefix 输出目录和前缀
五、结果（for newest version4）
# 输入文件
-rw-r--r-- 1 bayegy WST 13911342438 8月 4 11:00 CONT1_R1.fastq
-rw-r--r-- 1 bayegy WST 13911342438 8月 4 10:58 CONT1_R2.fastq
# 输出文件
tree non_rrna/
non_rrna/
├── cont1_fwd.fastq
└── cont1_rev.fastq
-rw-rw-r-- 1 cheng WST 13903074018 8月 27 18:24 cont1_fwd.fastq
-rw-rw-r-- 1 cheng WST 13903074018 8月 27 18:24 cont1_rev.fastq
